This page was written by Matt Ellis '98

This is a diagram of a Fluorescence Activated Cell Sorter
(FACS)

This image was obtained from a web site that
is no longer available <www9uchu.edu/grad/immuno/pizzo/how.htm>

Figure #3: The FACS allows cells (lymphocytes) to be separated and isolated
through the use of tagged antibodies or anti-immunoglobulins. This cartoon
representation shows the major hardware of FACS, its layout, and the basic
mechanisms. The sample mixture at the top of the figure is funneled through
a nozzlé which isolates cells and charges them. Cells pass from the
top of the figure towards the bottom. At the base of the FACS, there are
2 variably charged deflection plates and cell collectors (Lefrancois, Olson,
and Pizzo; http://www9uchu.edu/grad/immuno/pizzo/how.htm).

Sample Diluting System: in order to carry out FACS, the individual
cells in a resting lymphocyte pool must be isolated. This is accomplished
by suspending the cells in a fluid (i.e., saline). This suspension of cells
is then forced through a fine, high-pressure nozzle or fluidic diluting
system which distributes the cells into a single-file line or flow cell
(Kidd and Nicholson 229).

Light Source: light is an integral part of the FACS technique.
Light striking each cell is scattered. By using electronic devices that
measure scattered light and fluorescence, different types of cells and their
sizes can be identified. In older FACS devices, the preferred light source
was a mercury arc lamp. The more recent development of lasers has made the
mercury arc lamp somewhat obsolete. Laser light has several advantages:
it has a low amount of divergence, a high degree of intensity, and it can
be adjusted to provide a variety of very specific wavelengths of light (Kidd
and Nicholson 229).

Data Collecting Devices: FACSs have two types of data collecting
hardware: light scatter sensors and photomultiplier tubes (PMTs). The light
scatter sensors measure the light that is scattered by each cell from two
different angles. The forward angle light scatter sensor (FALS) gathers
light scattered in the forward direction. This type of scattered light gives
a clue as to a cell's size. Right angle, orthogonal, or side light scatter
(SS) sensors gather light scattered at 90° from the original direction
of the light source. This light reveals cell granularity, refractiveness,
and the presence of intracellular structures that reflect light (Darzynkiewicz,
et al. 335). Scatter sensors are useful in distinguishing cells based on
the cells' different structures. Neutrohphiles, for example, display more
SS than lymphocytes (Kidd and Nicholson 229). In addition, different cell
lineages or cells at different stages of development (i.e., pre B cells
versus plasma B cells) can be distinguished based on their forward and side
scatters (Kantor, Merrill, and Hillson 13.1). Finally, PMTs detect fluorescent
emissions from the fluorescent dyes on antibodies bound to cells or from
auto-fluorescence of the cells.

Integration Computer: this part of the FACS hardware is essential for
collecting information from the data collecting devices and integrating
it. The integrated information will be used in the formation of an electric
field that will select out cells that meet certain scatter and fluorescent
criteria.

Charged Plates: to collect selected cells, the cells are passed through
an electric field generated by oppositely-charged plates. By changing the
direction of the electric field between the plates, selected cells can be
directed into precise collection areas.